This project is directed towards determining a molecular basis for the modulation of G protein-coupled receptor signaling by polyunsaturated phospholipids, in particular those containing 22:6n-3 acyl chains. The membranes of neuronal and retinal tissue are unusually high in phospholipids containing one or two long chain polyunsaturated acyl chains. Docosahexaenoic acid (abbreviated DHA or 22:6n-3) is the major polyunsaturated acyl chain in these tissues. Studies are also focused on the role polyunsaturated phospholipids play in the response of receptors and signaling proteins to acute exposure. This year we initiated a study of phospholipids with very long (> 24 carbons) polyunsaturated acyl chains and refined previous studies of the differences between n-6 and n-3 polyunsaturated fatty acids (PUFAs).[unreadable] Phospholipids with extremely long chain polyunsaturated fatty acids (ELCPs) consisting of 26 to 36 carbons are found in excitatory tissues including rod outer segment disk membranes. In rod outer segment disk membranes they comprise at least 10% of the PC species. The possible functional roles of these ELCPs are unknown. The goal of this study was to determine the manner in which ELCPs alter membrane stability and acyl chain packing and determine the effects of ELCPs on receptor function and G protein-coupled signal transduction. Normal phase HPLC was used to isolate a PC fraction of disk membrane phospholipids that was more than 95% ELCP. Preliminary molecular characterization of this ELCP-PC fraction with capillary MS/MS shows that 50% of the acyl chains are 22:6n-3 ~30% are 32 carbon species and ~15% are 34 carbon species. This ELCP-PC was mixed with varying amounts of 18:0,18:1PC in pure lipid vesicles and reconstituted with rhodopsin, transducin and phosphodiesterase (PDE). Rhodopsin activation, transducin binding, PDE activity, acyl chain packing, and both membrane and protein thermal stability were examined. In pure lipid bilayers increasing levels of ELCP-PC slightly disordered acyl chain packing and reduced the thermal stability of 18:0,18:1PC. Increasing levels of ELCP-PC greatly enhanced formation of the active MII state of rhodopsin. However, 40% ELCP-PC in 18:0,18:1PC lowered PDE activity by 50% at physiological levels of stimulus. Large increases in rhodopsin activation coupled with modest relaxation in acyl chain packing suggest that ELCP-PC promotes rhodopsin activation via direct interaction rather than via changes in acyl chain packing. The reduction in PDE activity in spite of increased rhodopsin activation at high levels of ELCP-PC suggests that optimal functioning of GPCR signaling may require an optimal level of ELCP. [unreadable] In the retinal rod outer segment 40% to 50% of the phospholipid acyl chains consist of docosahexaenoic acid (DHA, 22:6n-3). Diets that are deficient in n-3, or u-3, fatty acids lead to the replacement of 22:6n-3 with 22:5n-6. Such diets also lead to suboptimal function in the neural pathways associated with learning, memory, intelligence, and visual function. We determined that the differences in ensemble acyl chain packing in bilayers consisting of 18:0,22:6n-3 PC, 18:0,22:5n-6 PC, and 18:0,22:5n-3 PC result in changes in receptor function and G protein-coupled signal transduction. The individual steps in visual signal transduction; receptor activation, G protein binding, effector (phosphodiesterase, PDE) activity and acyl chain packing were examined in membranes consisting of phosphatidylcholines with sn-1 = 18:0, and sn-2 = 22:6n-3 or 22:5n-6 or 22:5n-3 reconstituted with the appropriate membrane proteins. Fluorescent probe motion was the most rapid in 22:6n-3 and probe ensemble order was the lowest in the two n-3 PUFAs. The formation of both the activated MII state of rhodopsin and the MII-Gt complex was much slower in 18:0,22:5n-6 PC than in 18:0,22:6n-3 PC. In 18:0,22:5n-6 PC. PDE activity is only 60% of that observed in either 18:0,22:6n3 PC or 18:0,22:5n3 PC. These results establish that GPCR function is sensitive to changes in bond configuration of 22-carbon fatty acids at the phospholipid sn-2 position. In addition, particular molecular interactions that are vital to the ubiquitous G protein-coupled signaling mechanism are compromised by the replacement of 22:6n-3 by 22:5n-6.